Changes between Version 2 and Version 3 of SOPs/variant_calling

Timestamp:

10/15/13 14:40:17 (12 years ago)

Author:

gbell

Comment:

--

SOPs/variant_calling

@@ -5 +5 @@
 * mapping short reads
 * calling raw variants
-* filtering (really more like 'tagging') variants
-* annotating variants
+* adding filters (really more like 'tagging') and some annotations to variants
+* annotating effect(s) of variants on genes
 
 == Map short reads ==
@@ -43 +43 @@
 }}}
 
-== Call raw variants ==
+== Call raw variants with mpileup+bcftools ==
+
+Call variants (one sample vs. reference) with samtools' mpileup+bcftools (see the [http://samtools.sourceforge.net/mpileup.shtml| samtools' variant calling page] for more details)
+{{{
+samtools mpileup -d100000 -uf /nfs/genomes/sgd_2010/bwa/sacCer3.fa A_reads.bt2.sorted_unique.bam | bcftools view -bvcg - >| A_reads.bt2.sorted_unique.raw.bcf
+}}}
+Call variants (multiple sample vs. reference) using a set of BAM files
+{{{
+samtools mpileup -d100000 -uf /nfs/genomes/sgd_2010/bwa/sacCer3.fa *_reads.bt2.sorted_unique.bam | bcftools view -bvcg - >| ALL_reads.bt2.sorted_unique.raw.bcf
+}}}
+
+== Add filters and annotations to raw variants ==
+
+This step uses vcf-annotate from the [http://vcftools.sourceforge.net/docs.html VCFtools suite]
+
+Annotate variants by adding tags ("filters" but all variants are kept) to each variant, using all default filters
+{{{
+bcftools view -L -vcg A_reads.bt2.sorted_unique.raw.bcf | vcf-annotate -f + > A_reads.bt2.sorted_unique.withTags.bcf
+}}}
+
+Prepare file of known SNPs for use with vcf-annotate.
+Start with tab-delimited file (ex: SNP137.bed) that looks like 
+
+chr1    1360    1361    rs000000001
+{{{
+bgzip SNP137.bed
+tabix -p bed SNP137.bed.gz
+}}}
+
+Annotate variants by adding tags, more analysis, and any SNPdb overlaps
+{{{
+bcftools view -L -vcg A_reads.bt2.sorted_unique.raw.bcf | vcf-annotate -f +/d=10 --fill-HWE --fill-type -n -a SNP137.bed.gz -c CHROM,FROM,TO,INFO/SNP_ID -d key=INFO,ID=SNP_ID,Number=1,Type=Integer,Description='SNP137 sites' > A_reads.bt2.sorted_unique.filtered.vcf
+}}}
+
+Any tags will appear in the FILTER field, and SNPdb overlaps will appear in the INFO field of the output VCF file.
+
+Overlap with annotations can also be identified with intersectBed (where annotation will appear in new fields of the output VCF file):
+{{{
+intersectBed -wao -split -a A_reads.bt2.sorted_unique.raw.vcf -b SNP137.bed > A_reads.bt2.sorted_unique.annotated.vcf
+}}}
+
+== Annotate effect(s) of variants on genes ==
+
+Use snpEff (assuming snpEff has gene+protein annotations for your genome):
+{{{
+snpEff -c /usr/local/share/snpEff/snpEff.config -s A_snpEff.html SacCer_Apr2011.18 A_reads.bt2.sorted_unique.filtered.vcf > A_reads.bt2.sorted_unique.filtered.snpEff.vcf 
+}}}
+
+Get information only for variants that overlap protein-coding exons:
+{{{
+snpEff -c /usr/local/share/snpEff/snpEff.config -no-downstream -no-intergenic -no-intron -no-upstream -no-utr -s A_snpEff.html SacCer_Apr2011.18 A_reads.bt2.sorted_unique.filtered.vcf > A_reads.bt2.sorted_unique.filtered.snpEff.vcf
+}}}